Melanogenesis is the process of production and subsequent distribution of melanin by melanocytes within the skin and hair follicles [1, 2]. Melanocytes have specialized lysosome-like organelles, termed melanosomes, which contain several enzymes that mediate the production of melanin. The copper-containing enzyme tyrosinase catalyzes the oxidation of the amino acid tyrosine into DOPA and subsequently DOPA-quinone. At least two additional melanosomal enzymes are involved in the eumelanogenesis pathway that produces brown and black pigments, including TRP-1 (DHICA oxidase), and TRP-2 (DOPAchrome tautomerase). Depending on the incorporation of a sulfur-containing reactant (e.g. cysteine or glutathione) into the products, the melanogenesis pathway diverges to produce pheomelanins (amber and red pigments).
The perceived color of skin and hair is determined by the ratio of eumelanins to pheomelanins, and in part on blood within the dermis. The balance in skin hue is genetically regulated by many factors, including but not limited to: (a) the levels of expression of tyrosinase, TRP-2, and TRP-1; (b) thiol conjugation (e.g. with glutathione or cysteine) leading to the formation of pheomelanins; (c) the α-melanocyte-stimulating hormone (α-MSH) and melanocortin receptor, which is coupled to the adenylate cyclase/protein kinase A pathway; [15] (d) the product of the agouti locus, agouti signal protein, which has been documented to down-regulate pigmentation of hair melanocytes in rodents; [16] and (e) yet unknown mechanisms that regulate the uptake and distribution of melanosomes in recipient epidermal and hair matrix keratinocytes. [2, 13, 14]
Abnormalities of human skin pigmentation occur as a result of both genetic and environmental factors. Exposure of skin (especially Caucasian) to ultraviolet radiation, particularly in the UVB (i.e. intermediate) wavelengths, upregulates synthesis of melanocyte tyrosinase resulting in increased melanogenesis and thus tanning. However, acute or persistent UVB exposure can result in the formation of hyperpigmented lesions or regions of skin, including malignant melanoma skin cancer. [17] Both actinic damage and constitutional abnormalities can produce affected regions such as melasma, age spots, liver spots, freckles and other lentigenes. [3, 18, 19]
Vitiligo is the converse of hyperpigmentation, in which cutaneous melanocytes are either ablated or fail to produce sufficient pigment. [17, 18, 20] Although it would be desirable to restore lost pigmentation in vitiligo-affected skin with topical therapies, this has proven to be quite difficult to accomplish in a high proportion of subjects. As an alternative to PUVA therapy or cosmetic camouflage with dihydroxyacetone sunless-tanning lotions, [18] one might reduce the normal pigmentation of the unaffected skin to reduce contrast. Furthermore, a global market demand has developed for skin-lightening agents as “vanity” cosmeceutical products, because lighter skin color is preferred by some dark-skinned individuals in many countries and races, for psychological or sociological reasons. [4, 5]
Some purportedly “active” or “functional” agents for lightening skin color (e.g. arbutin, kojic acid, niacinamide, licorice, magnesium ascorbyl phosphate, among others) have not been demonstrated yet to be clinically efficacious when critically analyzed in carefully controlled studies [5, 6, 25]. The U.S. FDA-approved pharmaceutical products containing 2-4% hydroquinone (“HQ”) are minimally to moderately efficacious. However, HQ has been demonstrated to be cytotoxic to cultured mammalian melanocytes, and mutagenic in Salmonella and mammalian Chinese hamster V79 cells [3-6, 10, 11, 25]. HQ appears to be an important intermediate in the bioactivation of the carcinogen benzene [12]. Although it has been repeatedly asserted in the dermatologic literature for many years, without substantiation, that HQ is an inhibitor of tyrosinase, this compound is not an effective inhibitor of the mammalian enzyme [5, 6, 25]. Hydroquinone's in vitro mechanism of action appears to be primarily a melanocytic cytotoxic effect. Its clinical mechanism of action on whole skin remains uncertain.
In view of these biochemical disadvantages of the standard skin bleaching agent, HQ, it is highly desirable to identify other compounds with improved efficacy and safety characteristics. Methyl gentisate (“MG”), the methyl ester of gentisic acid (GA; 2,5-dihydroxybenzoic acid), is a moderately potent inhibitor of melanin accumulation in a murine melanocyte cell culture primary screen [6, 25]. GA is a natural product from the root of the genus Gentiana, named after Gentius, an Illyrian (Greco-Roman) king of the 2nd century B.C., said to have first discovered the medicinal properties of the plant [7]. The sodium salt of GA is thought to be an analgesic and an anti-inflammatory agent. GA is a ubiquitous metabolite, produced not only by plants, but also by Penicillium patulum and Polyporus tumulosus, and is excreted into the urine of mammals following ingestion of salicylates [8, 9]. MG and GA are simple phenolic compounds structurally similar to HQ, yet lacking the mutagenic activity of HQ [25]. MG has not been developed as a commercially available topical skin lightener product to date.
Two patent publications of Sansei Seiyaku also disclose a number of compounds, which allegedly are active as either tyrosinase inhibitors or as skin lightening agents, JP 5-124925 and JP 5-124922. The compounds are various benzimidazolethiols, but have not been developed as commercially available topical skin lightener products to date. In addition, phenylthiourea (PTU) has been reported as an inhibitor of tyrosinase, but has not been developed as a commercially available topical skin lightener product to date [30-32].
It is an object of this invention to provide methods and compositions for reducing pigmentation in skin from mammals, including humans.
Another object is to provide methods and compositions for reducing pigmentation of skin for cosmetic, beauty-enhancing, or esthetic effects.
It is another object to provide methods and compositions for treating hyperpigmentation-related medical conditions such as melasma, age spots, freckles, ochronosis, postinflammatory hyperpigmentation, lentigo, and other pigmented skin blemishes.
Another object of the present invention is to provide methods and compositions for inhibiting mammalian melanocyte tyrosinase, the rate-limiting enzyme in the production of melanin from tyrosine and DOPA.
Still another object of the invention is to provide methods and compositions to absorb ultraviolet radiation (UVR), and thus to protect skin from UVR and photoaging.
An additional object of the invention is to provide antioxidant compositions that protect skin from oxidative damage, and/or to prevent oxidative decomposition of product formulations.
Another object is to facilitate discovery of compounds that inhibit mammalian tyrosinase in cell-free extracts from mammalian melanocyte or melanoma cells, using either a colorometric DOPA oxidation or a radiolabeled tyrosine or DOPA substrate assay (IC50≦300 μM).
Another object is to facilitate discovery of compounds that inhibit de novo pigment production (synthesis and/or accumulation) in cultured mammalian melanocyte or melanoma cells (IC50≦300 μM).
Another object is to facilitate evaluation of compounds for toxicity in mammalian melanocyte, melanoma, or other cell cultures (IC50≦300 μM.
Another object is to provide composition of matter and/or identity of compounds that are efficacious and/or exhibit reduced toxicity using one or more of the bioassays described in other objects, with biochemical characteristics equivalent to or superior to hydroquinone or methyl gentisate.
Another object is to provide active and/or functional compounds from diverse structural classes, including but not limited to the following examples: benzoimidazoles, phenylamines, phenylthioureas, phenols, and phenylthiols.
Still another object is to provide synthesis of derivatives of active and/or functional compounds of the invention, including by organic synthesis, combinatorial chemistry, medicinal chemistry, X-ray crystallography, rational drug design, and other methods.
Another object is to provide the use of formulations of the present invention for cosmetic, cosmeceutical, over-the-counter drug, and prescription drug products.
Another object is to provide formulations of the present invention for the purpose of reducing or preventing pigmentation in hair, albeit during the biosynthesis of hair, as a result of blocking pigment production within the melanocytes of hair follicles.
Another object is to provide the active and/or functional compounds of the present invention for use in inhibiting tyrosinase or tyrosinase-like enzymes from non-mammalian species, for instance for use in the food science industry for the inhibition of enzymatic browning.
Still another object is to provide the active and/or functional compounds of the present invention for use in inhibiting tyrosine hydroxylase enzymes, in order to reduce the biosynthesis of DOPA and/or catecholamines.